The airway epithelium may be damaged extensively in inflammatory responses to environmental agents and allergen exposure, in part by apoptosis (programmed cell death). One cytokine, transforming growth factor-beta (TGF-beta), may prevent epithelial cell apoptosis. Given the role of TGF-beta in mediating both inflammation and tissue remodeling, understanding the mechanisms by which TGF-beta may protect airway epithelium is of practical importance. We will examine mechanisms by which TGF-beta may regulate airway epithelial cell apoptosis in three specific aims. Aim #1: identify mechanisms by which TGF-beta regulates airway epithelial cell apoptosis. We identify key regulators in the Smad pathway that mediate the anti-apoptotic effect of TGF-beta in primary epithelial cells and cell lines in which death is induced by ligation of the Fas (CD95) receptor. We will examine this protective effect is blocked by the key inhibitor Smad7 and whether this protective effect is mediated via activation of nuclear transcription factors such as Sp1. We will identify potential, counter-balancing pro-apoptotic pathways mediated by either p38 MAPK or JNK that may be activated by TGF-beta. We will examine whether growth arrest mediates the TGF-beta mediated anti-apoptotic effect in airway epithelium and whether driving cells past G1 growth arrest by over-expression of cyclin D1 or cyclin E ablates the protective effect of TGF-beta. We will identify how key cell cyclin dependent kinases (CDK) 2 and 4, and the activity of their inhibitors p21 waf1 and p15Ink4B, modulate the interplay between growth arrest and apoptosis. Aim #2: identify how TGF-beta modulates airway epithelial cell apoptosis using models of apoptosis and airway inflammation. We will use models of acute allergen-induced airway inflammation, and of Fas activation, in mice in which epithelial cell shedding and apoptosis occurs to examine whether exogenous TGF-beta can reverse epithelial cell apoptosis, and whether conditional over-expression of TGF-beta can prevent and reverse epithelial cell shedding and death by either allergen or by Fas activation. Aim #3: identify the role of TGF-beta in normal subjects and subjects with persistent asthma. We will grow airway epithelium collected from asthmatic and normal subjects and examine differences in the protective effect of TGF-beta after Fas ligation. We will identify differences in cell cycle, p21 and p15 expression, and cell activation that may explain the relative resistance of asthmatic epithelial cells to the protective effect of TGF-beta. Together, these studies should provide important new insights into a fundamental mechanism controlling airway epithelial homeostasis. Understanding this process may suggest new therapeutic interventions to protect the airway epithelium from damage in asthma.